Abstract:

A probe, a set of probes, and a probe carrier on which the probe or the
set of probes is immobilized, are provided for classification of fungus
species. The probe or the set of probes is capable of collectively
detecting fungus of the same species and distinguishingly detecting those
fungus from fungus of other species. The probe is an oligonucleotide
probe for detecting a pathogenic fungus DNA and includes at least one of
base sequences of SEQ ID NOS. 1 to 4 and mutated sequences thereof.

Claims:

1. A probe for detecting a DNA of Candida glabrata which is a pathogenic
fungus, comprising one of the following base sequences (1) to (5):(1)
ggtgttttatcacacgactcgacact (SEQ ID NO. 1) or a complementary sequence
thereof;(2) ggagttctcccagtggatgcaaac (SEQ ID NO. 2) or a complementary
sequence thereof;(3) ggccatatcagtatgtgggacacg (SEQ ID NO. 3) or a
complementary sequence thereof;(4) aggttttaccaactcggtgttgatctag (SEQ ID
NO. 4) or a complementary sequence thereof; and(5) a mutated sequence
which is obtained by deletion, substitution, or addition of a base on one
of the sequences of SEQ ID NOS. 1 to 4 and the complementary sequences
thereof within a range that the mutated sequence retains a function as
the probe.

2. A probe set for detecting a DNA of Candida glabrata which is a
pathogenic fungus, comprising at least two of the following probes (A) to
(P):(A) a probe including a base sequence represented by
ggtgttttatcacacgactcgacact (SEQ ID NO. 1);(B) a probe including a base
sequence represented by ggagttctcccagtggatgcaaac (SEQ ID NO. 2);(C) a
probe including a base sequence represented by ggccatatcagtatgtgggacacg
(SEQ ID NO. 3);(D) a probe including a base sequence represented by
aggttttaccaactcggtgttgatctag (SEQ ID NO. 4);(E) a probe including a
complementary sequence of SEQ ID NO. 1;(F) a probe including a
complementary sequence of SEQ ID NO. 2;(G) a probe including a
complementary sequence of SEQ ID NO. 3;(H) a probe including a
complementary sequence of SEQ ID NO. 4;(I) a probe including a mutated
sequence obtained by deletion, substitution or addition of a base on SEQ
ID NO. 1 within a range that the mutated sequence retains a function as
the probe;(J) a probe including a mutated sequence obtained by deletion,
substitution or addition of a base on SEQ ID NO. 2 within a range that
the mutated sequence retains the function as the probe;(K) a probe
including a mutated sequence obtained by deletion, substitution or
addition of a base on SEQ ID NO. 3 within a range that the mutated
sequence retains the function as the probe;(L) a probe including a
mutated sequence obtained by deletion, substitution or addition of a base
on SEQ ID NO. 4 within a range that the mutated sequence retains the
function as the probe;(M) a probe including a mutated sequence obtained
by deletion, substitution or addition of a base on the complementary
sequence of SEQ ID NO. 1 within a range that the mutated sequence retains
the function as the probe;(N) a probe including a mutated sequence
obtained by deletion, substitution or addition of a base on the
complementary sequence of SEQ ID NO. 2 within a range that the mutated
sequence retains the function as the probe;(O) a probe including a
mutated sequence obtained by deletion, substitution or addition of a base
on the complementary sequence of SEQ ID NO. 3 within a range that the
mutated sequence retains the function as the probe; and(P) a probe
including a mutated sequence obtained by deletion, substitution or
addition of a base on the complementary sequence of SEQ ID NO. 4 within a
range that the mutated sequence retains the function as the probe.

3. A probe carrier, comprising a plurality of probes which form the probe
set according to claim 2, wherein:the plurality of probes are each
immobilized on a carrier; andthe plurality of probes are arranged at
intervals.

4. A method of detecting a DNA of Candida glabrata in a sample by using a
probe carrier, comprising:(i) reacting the sample with the probe carrier
according to claim 3; and(ii) detecting a reaction intensity of a probe
on the probe carrier reacted with a nucleic acid contained in the sample.

5. A kit for detecting a DNA of Candida glabrata, comprising:the probe
according to claim 1; anda reagent for detecting a reaction of a probe
with a target nucleic acid.

6. A kit for detecting a DNA of Candida glabrata, comprising:a plurality
of probes in the probe set according to claim 2; anda reagent for
detecting a reaction of a probe with a target nucleic acid.

7. A kit for detecting a DNA of Candida glabrata, comprising:the probe
carrier according to claim 3; anda reagent for detecting a reaction of a
probe with a target nucleic acid.

Description:

BACKGROUND OF THE INVENTION

[0001]1. Field of the Invention

[0002]The present invention relates to a probe and a probe set for
detecting a DNA of a pathogenic fungus, Candida glabrata, which are
useful for detection and identification of a causative fungus of an
infectious disease, and to a probe carrier on which the probe or the
probe set is immobilized. The present invention also relates to a DNA
testing method and a DNA testing kit using the same.

[0003]2. Description of the Related Art

[0004]Heretofore, reagents for and methods of quickly and accurately
detecting pathogenic fungi in a sample have been proposed. For instance,
Japanese Patent Application Laid-Open No. H08-089254 discloses
oligonucleotides having specific base sequences, which can be used as
probes and primers for detecting pathogenic fungi of candidiasis and
aspergillosis, and a method of detecting target fungi using such
oligonucleotides. In addition, the same patent document also discloses a
primer set used for PCR amplifying a plurality of target fungi in common.
Further, the same patent document also discloses a method of identifying
fungus species in a sample comprising subjecting a plurality of target
fungi in the sample to PCR amplification using the primer set, and then
detecting the sequence portions specific to the respective fungi by a
hybridization assay using the probes specific to the respective fungi.

[0005]On the other hand, a method capable of simultaneously detecting a
plurality of oligonucleotides having different base sequences is known.
The method uses a probe array in which probes having sequences
complementary to the respective base sequences are arranged at intervals
on a solid phase (Japanese Patent Application Laid-Open No. 2004-313181).

SUMMARY OF THE INVENTION

[0006]However, it is not easy to establish a probe which specifically
detects a DNA of a pathogenic fungus in a sample. The sample may contain
not only the DNA of the pathogenic fungus but also DNAs of other
pathogenic fungi. Thus, it is not easy to establish a probe that
specifically detects a DNA of a pathogenic fungus which is less
susceptible to the influence of the presence of DNAs of other pathogenic
fungi (i.e. cross contamination). Under such circumstances, the inventors
of the present invention have conducted investigation on probes for
detecting the following pathogenic fungi. The object of the investigation
was to obtain probes capable of detecting a DNA of a target pathogenic
fungus with a high degree of accuracy even for an analyte containing DNAs
of a plurality of fungi with a less cross-contamination level. As a
result, a plurality of probes capable of detecting a pathogenic fungus
DNA with a high degree of accuracy were finally obtained.

[0007]Candida glabrata

[0008]A first object of the present invention is to provide a probe and a
probe set which are capable of accurately identifying the DNA of a target
fungus.

[0009]Another object of the present invention is to provide a probe
carrier which is capable of accurately identifying a target fungus from a
sample in which various kinds of fungi may exist together.

[0010]Still another object of the present invention is to provide a DNA
testing method for a pathogenic fungus, which can more quickly and more
accurately detect a target fungus from a sample when various kinds of
fungi exist in the sample, and to provide a kit for the testing method.

[0011]The probe of the present invention for detecting a DNA of Candida
glabrata which is a pathogenic fungus includes one of the following base
sequences (1) to (5):

[0016](5) a mutated sequence which is obtained by deletion, substitution,
or addition of a base on one of the sequences of SEQ ID NOS. 1 to 4 and
the complementary sequences thereof in a range that the mutated sequence
retains a function as the probe.

[0017]In addition, the probe set of the present invention for detecting a
DNA of Candida glabrata which is a pathogenic fungus includes at least
two of the following probes (A) to (P):

[0026](I) a probe including a mutated sequence obtained by deletion,
substitution or addition of a base on SEQ ID NO. 1 within a range that
the mutated sequence retains a function as the probe;

[0027](J) a probe including a mutated sequence obtained by deletion,
substitution or addition of a base on SEQ ID NO. 2 within a range that
the mutated sequence retains the function as the probe;

[0028](K) a probe including a mutated sequence obtained by deletion,
substitution or addition of a base on SEQ ID NO. 3 within a range that
the mutated sequence retains the function as the probe;

[0029](L) a probe including a mutated sequence obtained by deletion,
substitution or addition of a base on SEQ ID NO. 4 within a range that
the mutated sequence retains the function as the probe;

[0030](M) a probe including a mutated sequence obtained by deletion,
substitution or addition of a base on the complementary sequence of SEQ
ID NO. 1 within a range that the mutated sequence retains the function as
the probe;

[0031](N) a probe including a mutated sequence obtained by deletion,
substitution or addition of a base on the complementary sequence of SEQ
ID NO. 2 within a range that the mutated sequence retains the function as
the probe;

[0032](O) a probe including a mutated sequence obtained by deletion,
substitution or addition of a base on the complementary sequence of SEQ
ID NO. 3 within a range that the mutated sequence retains the function as
the probe; and

[0033](P) a probe including a mutated sequence obtained by deletion,
substitution or addition of a base on the complementary sequence of SEQ
ID NO. 4 within a range that the mutated sequence retains the function as
the probe.

[0034]The probe carrier of the present invention includes a carrier on
which the probe or each of the plurality of probes constituting the probe
set mentioned above is placed on a carrier.

[0035]In the probe carrier of the present invention, at least one of the
probes (A) to (P) is immobilized on a carrier, and when a plurality of
probes is to be immobilized, the respective probes are placed while being
isolated from each other.

[0036]The method of detecting DNA of Candida glabrata in a sample by using
a probe carrier according to the present invention includes:

[0037](i) reacting the sample with the probe carrier; and

[0038](ii) detecting a reaction intensity of a probe on the probe carrier
reacted with a nucleic acid contained in the sample.

[0039]The kit for detecting a pathogenic fungus of the present invention
is a kit for detecting a DNA of Candida glabrata, including at least one
of the probes (A) to (P) or a probe carrier on which at least one of
those probes is immobilized and a reagent for detecting a reaction of a
probe with a target nucleic acid.

[0040]According to the present invention, when a specimen is infected with
the causative fungus mentioned above, the fungus can be more quickly and
precisely identified from the specimen even if the specimen is
simultaneously and complexly infected with other fungi in addition to the
above-mentioned fungus. In particular, Candida glabrata can be detected
while precisely distinguishing it from any of fungi of other Candida
species, fungi of Trichosporon species, fungi of Cryptococcus species,
fungi of Aspergillus species, fungi of Epidermophyton species, fungi of
Arthroderma species, and fungi of Trichophyton species, which may be more
likely to cause cross contamination.

[0041]Further features of the present invention will become apparent from
the following description of exemplary embodiments with reference to the
attached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0042]FIG. 1 is a diagram illustrating a 1st PCR protocol.

[0043]FIG. 2 is a diagram illustrating a 2nd PCR protocol.

[0044]FIG. 3 is a diagram illustrating a protocol of hybridization.

DESCRIPTION OF THE EMBODIMENTS

[0045]The present invention provides an oligonucleotide probe for
identifying a pathogenic fungus (hereinafter, simply referred to as a
probe) and a probe set including a combination of two or more probes. The
use of such a probe or a probe set allows the detection of the following
fungus which will cause inflammation by infection.

[0046](Fungus Name)

[0047]Candida glabrata

[0048]The probe of the present invention can react with the DNA sequence
of an internal transcribed spacer (ITS) of the pathogenic fungus or a
nucleic acid having a base sequence specific to the ITS region contained
in a sample. The probe can be selected from the oligonucleotides having
the following base sequences:

[0053](E) a probe including a complementary sequence of the base sequence
represented by SEQ ID NO. 1;

[0054](F) a probe including a complementary sequence of the base sequence
represented by SEQ ID NO. 2;

[0055](G) a probe including a complementary sequence of the base sequence
represented by SEQ ID NO. 3;

[0056](H) a probe including a complementary sequence of the base sequence
represented by SEQ ID NO. 4;

[0057](I) a probe including a mutated sequence obtained by deletion,
substitution or addition of a base on SEQ ID NO. 1 in a range that the
mutated sequence retains a function as the probe;

[0058](J) a probe including a mutated sequence obtained by deletion,
substitution or addition of a base on SEQ ID NO. 2 in a range that the
mutated sequence retains the function as the probe;

[0059](K) a probe including a mutated sequence obtained by deletion,
substitution or addition of a base on SEQ ID NO. 3 in a range that the
mutated sequence retains the function as the probe;

[0060](L) a probe including a mutated sequence obtained by deletion,
substitution or addition of a base on SEQ ID NO. 4 in a range that the
mutated sequence retains the function as the probe;

[0061](M) a probe including a mutated sequence obtained by deletion,
substitution or addition of a base on SEQ ID NO. 1 in a range that the
mutated sequence retains the function as the probe;

[0062](N) a probe including a mutated sequence obtained by deletion,
substitution or addition of a base on the complementary sequence of SEQ
ID NO. 2 in a range that the mutated sequence retains the function as the
probe;

[0063](O) a probe including a mutated sequence obtained by deletion,
substitution or addition of a base on the complementary sequence of SEQ
ID NO. 3 in a range that the mutated sequence retains the function as the
probe; and

[0064](P) a probe including a mutated sequence obtained by deletion,
substitution or addition of a base on the complementary sequence of SEQ
ID NO. 4 in a range that the mutated sequence retains the function as the
probe.

[0065]The probe set can be prepared using two or more of those probes. The
ITS region of DNA of the pathogenic fungus can be sufficiently detected
using the probe set of the present invention.

[0066]The mutated sequences include any mutation as far as it does not
impair the function of the probe, or any mutation as far as it hybridizes
with a nucleic acid sequence of interest as a detection target. Of those,
it is desirable to include any mutation as far as it can hybridize with a
nucleic acid sequence of interest as a detection target under stringent
conditions. Preferable hybridization conditions confining the mutation
include those represented in examples as described below. Here, the term
"detection target" used herein may be one included in a sample to be used
in hybridization, which may be a unique base sequence to the pathogenic
fungus, or may be a complementary sequence to the unique base sequence.
Further, the variation may be a mutated sequence obtained by deletion,
substitution, or addition of at least one base as far as it retains
function as the probe.

[0067]The characteristics of those probes depend on the specificities of
the respective probe sequences to the target nucleic acid sequence as a
test object. The specificity of the probe sequence can be evaluated from
the degree of coincidence between the base sequence thereof and the
target nucleic acid sequence and the melting temperature of a duplex of
the target nucleic acid sequence and the probe sequence. In addition,
when the probe is constituting a probe set, the performance of the probe
also depends on a difference between the melting temperature of the probe
and the melting temperature of another probe sequence in the probe set.

[0068]For designing these probe sequences, base sequences with high
specificities to the same fungus species but with less variation between
different strains in the same species may be selected. In addition, a
region can be selected to have three or more unmatched bases with respect
to the sequence of the fungus species other than the fungus species of
interest. Further, it is designed such that the difference between the
melting temperature of the duplex of the probe sequence and the sequence
of the fungus species of interest and the melting temperature of the
duplex of the probe sequence and the sequence of any fungus species other
than the species of interest is 10° C. or more. Further, it is
designed such that any base is deleted from or added to the highly
specific region to hold the melting temperatures of the respective probes
immobilized on the same carrier within a predetermined temperature range.

[0069]The experiment conducted by the inventors of the present invention
has revealed that the attenuation of hybridization intensity may be small
when 80% or more of the continuous base sequence is conserved. Therefore,
of the probe sequence disclosed in the present application, any mutated
sequence may retain the functions of the probe as long as the mutated
sequence conserves 80% or more of the continuous base sequence.

[0070]Those probe sequences are specific to the DNA sequence of the ITS
region of the fungus of interest, so a sufficient hybridization
sensitivity to the sequence is expected. In addition, those probe
sequences are designed so that it will bring a good result by forming a
stable hybrid in a hybridization reaction with a target specimen even in
a state of being immobilized on a carrier. Further, those probes are
designed so that the melting temperature thereof falls within a
predetermined temperature range.

[0071]Further, those probe sequences are designed so that fungus species
can be determined with a combination of specific portions without
determining the sequence of the entire ITS region.

[0072]Further, a probe carrier (e.g., DNA chip), on which the probe for
detecting a pathogenic fungus of the present invention is immobilized,
can be obtained by supplying the probe on a predetermined position on the
carrier and immobilizing the probe thereon. Various methods can be used
for the supply of probe to the carrier. For example, a method which can
be suitably used is to keep the probe in a state of being immobilized on
the carrier through a chemical bonding (e.g., covalent bonding) and a
liquid containing the probe is then provided on a predetermined position
by an inkjet method. Such a method allows the probe to be hardly detached
from the carrier and exerts an additional effect of improving the
sensitivity. In other words, when a stamping method conventionally called
a Stanford method in common use is employed to make a DNA chip, the
resultant DNA chip has a disadvantage in that the applied DNA tends to be
peeled off. In addition, one of the methods of producing DNA chips is to
carry out the placement of probes by the synthesis of DNA on the surface
of a carrier (e.g., Oligonucleotide array manufactured by Affymetrix Co.,
Ltd.). In the method of synthesizing the probe on the carrier, it is
difficult to control synthesis amount for each probe. Thus, the amount of
immobilized probe per immobilization area (spot) for each probe tends to
vary considerably from one another. Such variations in amounts of the
respective immobilized probes may cause inaccurate evaluation on the
results of the detection with those probes. Based on this fact, the probe
carrier of the present invention is preferably prepared using the inkjet
method. The inkjet method as described above has an advantage in that the
probe can be stably immobilized on the carrier and hardly detaching from
the carrier to efficiently provide a probe carrier which can be detected
with high sensitivity and high accuracy.

[0073]For using a plurality of probes immobilized on a carrier, the probes
are designed to have a given melting temperature to simplify the protocol
of hybridization.

[0074]Hereinafter, preferred embodiments of the present invention will be
described in further detail with reference to the attached drawings.

[0075]First, the terms used herein will be elucidated as follows:

[0076]The term "specimen" represents one obtained as a target of an
examination. The term "analyte" represents one prepared from the analyte
to contain DNA or nucleic acid fragments. The term "sample" represents a
target to be reacted with a probe. The "sample" includes the specimen
when the specimen is directly reacted with a probe. The "sample" includes
the analyte when the analyte prepared from the specimen is directly
reacted with a probe.

[0077]The specimen to be tested using probe carriers (e.g., DNA chips) in
which the probes of the present invention are immobilized on carriers
include those originated from humans and animals such as domestic
animals. For example, the test object is any of those which may contain
fungi including: any body fluids such as blood, cerebrospinal fluid,
expectorated sputum, gastric juice, vaginal discharge, and oral mucosal
fluid; tissues such as skin, fingernails, and hair; and excretions such
as urine and feces. All media, which can be contaminated with fungi, can
be also subjected to a test using the DNA chip. The media include: food,
drink water, and water in the natural environment such as hot spring
water, which may cause food poisoning by contamination; filters of air
cleaners and the like; and the like. Animals and plants, which should be
quarantined in import/export, are also used as specimens of interest.

[0078]When the specimen as described above can be directly used in
reaction with the DNA chip, it is used as a sample to react with the DNA
chip and the result of the reaction is then analyzed. Further, when the
specimen cannot be directly reacted with the DNA chip, the analyte was
subjected to extraction, purification, and other procedures for obtaining
a target substance as required and then provided as a sample to carry out
a reaction with the DNA chip. For instance, when the specimen contains a
target nucleic acid, an extract, which may be assumed to contain such a
target nucleic acid, is prepared from a specimen, and then washed,
diluted, or the like to obtain an analyte as a sample followed by
reaction with the DNA chip. Further, when a target nucleic acid is
included in a specimen obtained by carrying out various amplification
procedures such as PCR amplification, the target nucleic acid may be a
sample for being amplified and then reacted with a DNA chip. Such
analytes of amplified nucleic acids include the following.

[0079](a) An amplified analyte prepared by using a PCR-reaction primer
designed for detecting ITS region.

[0080](b) An amplified analyte prepared by an additional PCR reaction or
the like from a PCR-amplified product.

[0081](c) An analyte prepared by an amplification method other than PCR.

[0082](d) An analyte labeled for visualization by any of various labeling
methods.

[0083]Further, a carrier used for preparing a probe carrier such as a DNA
chip may be any of those that satisfy the property of carrying out a
solid phase-liquid phase reaction of interest. Examples of the carrier
include: flat substrates such as a glass substrate, a plastic substrate,
and a silicon wafer; a three-dimensional structure having an irregular
surface; a spherical body such as a bead; and rod-, cord-, and
thread-shaped structures. The surface of the carrier may be processed
such that a probe can be immobilized thereon. In particular, a carrier
prepared by introducing a functional group to its surface to make
chemical reaction possible has a preferable form from the viewpoint of
reproducibility because the probe is stably bonded in the process of
hybridization reaction.

[0084]Various methods can be employed for the immobilization of probes. An
example of the method of using a combination of a maleimide group and a
thiol (--SH) group. In this method, a thiol (--SH) group is bonded to the
terminal of a probe, and a process is executed in advance to make the
carrier (solid phase) surface have a maleimide group. Accordingly, the
thiol group of the probe supplied to the carrier surface reacts with the
maleimide group on the carrier surface to form a covalent bond, whereby
the probe is immobilized.

[0085]First, introduction of the maleimide group can utilize a process of
allowing a reaction of a glass substrate with an aminosilane coupling
agent and then introducing an maleimide group onto the glass substrate by
a reaction of the amino group with an EMCS reagent
(N-(6-maleimidocaproyloxy)s--ccinimide, manufactured by Dojindo).
Introduction of the thiol group to a DNA can be carried out using
5'-Thiol-Modifier C6 (manufactured by Glen Research) when the DNA is
synthesized by an automatic DNA synthesizer. Instead of the combination
of a thiol group and a maleimide group, a combination of, e.g., an epoxy
group (on the solid phase) and an amino group (nucleic acid probe
terminal), can also be used as a combination of functional groups to be
used for immobilization. Surface treatments using various kinds of silane
coupling agents are also effective. A probe in which a functional group
which can react with a functional group introduced by a silane coupling
agent is used. A method of applying a resin having a functional group can
also be used.

[0086]The detection of pathogenic fungus DNA using the probe carrier of
the present invention can be carried out by a DNA-detecting method at
least including:

[0087](i) reacting a sample with the probe carrier on which the probe of
the present invention is immobilized; and

[0088](ii) detecting the reaction intensity of the probe on the probe
carrier which is reacted with the nucleic acid contained in the sample.

[0089]In addition, the detection can be carried out by a detecting method
at least including:

[0090](a) reacting a sample with the probe carrier on which the probe of
the present invention is immobilized;

[0091](b) detecting the reaction intensity of the probe on the probe
carrier with a nucleic acid in the sample; and

[0092](c) specifying the probe reacted with the nucleic acid in the sample
when the reaction of the probe with the nucleic acid in the sample is
detected and specifying the DNA of a pathogenic fungus in the sample
based on the base sequence of the probe.

[0093]The probe to be immobilized on the probe carrier is at least one of
the above-mentioned items (A) to (P). On the carrier, other probes (those
for detecting fungus species other than Candida glabrata) may be
immobilized depending on the purpose of test. In this case, the other
probes may be those capable of detecting the fungus species other than
Candida glabrata without causing cross contamination and the use of such
probes allows simultaneous detection of a plurality of fungus species
with high accuracy.

[0094]As described above, when the base sequence of the ITS region on the
DNA sequence of the pathogenic fungus contained in the specimen being
amplified by PCR is used as a sample to be reacted with the probe
carrier, the primer set for detecting the pathogenic fungus can be used.
The primer set is preferably one containing oligonucleotides having the
following known base sequences:

[0095]Accordingly, the detection method of the present invention may
further include amplifying the target nucleic acid in the specimen by PCR
using oligonucleotides having the above-mentioned base sequences (1) and
(2) as primers.

[0096]A kit for detecting a pathogenic fungus can be prepared using at
least any of the probes as described above and a reagent for detecting a
reaction of the probe with nucleic acid in a sample. The probe in such a
kit may be provided as the probe carrier. In addition, the reagent for
the detection may contain a primer to be used in labeling for detecting
the detection or used in amplification as a pretreatment. In the case
where the detection reagent contains the primer, the primer is preferably
one suitable for amplifying the DNA of the ITS region of Candida
glabrata. Further, the detection reagent may contain a primer for
applying the DNA of the ITS region of any pathogenic fungus other than
Candida glabrata in addition to the primer for amplifying the DNA of the
ITS region of Candida glabrata.

EXAMPLES

[0097]Hereinafter, the present invention is described in further details
with reference to examples using probes for detecting pathogenic fungi.

Example 1

Production of DNA Chip

[0098]In this example, the production of a DNA chip on which the probe of
the present invention is immobilized is described.

[0099](1. Preparation of Probe DNA)

[0100]First, nucleic acid sequences represented in Table 1 were designed
as probes for detecting fungi. Specifically, the probe base sequences
represented as follows were selected from the ITS regions on the fungal
DNA sequences represented in Table 1. Those probe base sequences are
designed so that they will be expected to have extremely high
specificities and sufficient hybridization sensitivities without any
variation in the respective probe base sequences. Note that the probe
base sequences do not have to be limited to the sequences which
completely correspond to those represented in Table 1. The base sequences
having about 20 to 30 bases each of which include the probe base
sequences may be also included in the probe base sequences of Table 1. In
this case, other portions other than the base sequences as defined in
Table 1 should have base sequences which do not affect the detection
accuracy.

[0101]In each of the probes shown in Table 1, a thiol group was introduced
on the 5' terminal of the nucleic acid as a functional group to be
immobilized on a DNA chip in accordance with the conventional procedures.
After the introduction of the functional group, the probe was purified
and then freeze-dried. Subsequently, the freeze-dried probe was stored in
a refrigerator at -30° C.

[0102](2. Preparation of PCR Primer)

[0103](2-1. Preparation of PCR Primer for Specimen Amplification)

[0104]Conventional primers are used as PCR primers for amplifying the ITS
region on DNA to be used for detecting a pathogenic fungus. The
conventional primers are shown in Table 2. Specifically, the conventional
primers are provided as a set of conventional primers for specifically
amplifying the ITS region of DNA, designed from regions common to fungi
so as to carry out simultaneous amplification even when fungus species
are different.

[0105]The primers shown in Table 2 were synthesized and then purified by
high-performance liquid chromatography (HPLC). Subsequently, each primer
was dissolved in a TE buffer to obtain the primer at a final
concentration of 10 pmol/μl.

[0106](2-2. Preparation of PCR Primer for Labeling)

[0107]A primer for labeling is prepared by introducing a label into a
reverse primer of the primer for specimen amplification. The primer for
labeling is shown in Table 3.

[0108]The primer shown in Table 3 was labeled with fluorescent dye Cy3.
After the synthesis, the primer was purified by high-performance liquid
chromatography (HPLC) and then dissolved in a TE buffer to obtain the
primer at a final concentration of 10 pmol/μl.

[0109](3. Production of DNA Chip)

[0110](3-1. Cleaning of Glass Substrate)

[0111]A glass substrate (size: 25 mm×75 mm×1 mm, manufactured
by IIYAMA PRECISION GLASS CO., LTD.) formed of synthetic quartz was
placed in a heat- and alkali-resisting rack and immersed in a cleaning
solution for ultrasonic cleaning, which was adjusted to have a
predetermined concentration. The glass substrate was kept immersed in the
cleaning solution for a night and cleaned by ultrasonic cleaning for 20
minutes. The substrate was picked up, lightly rinsed with pure water, and
cleaned by ultrasonic cleaning in ultrapure water for 20 minutes. The
substrate was immersed in a 1N aqueous sodium hydroxide solution heated
to 80° C. for 10 minutes. Pure water cleaning and ultrapure water
cleaning were executed again. A quartz glass substrate for a DNA chip was
thus prepared.

[0112](3-2. Surface Treatment)

[0113]A silane coupling agent KBM-603 (manufactured by Shin-Etsu Chemical
Co., Ltd.0) was dissolved in pure water at a concentration of 1% by
weight (wt %) and stirred at room temperature for 2 hours. Subsequently,
the cleaned glass substrate was immersed in the aqueous solution of the
silane coupling agent and left standing at room temperature for 20
minutes. The glass substrate was picked up. The surface thereof was
lightly rinsed with pure water and dried by spraying nitrogen gas to both
surfaces of the substrate. The dried substrate was baked in an oven at
120° C. for 1 hour to complete the coupling agent treatment,
whereby an amino group was introduced to the substrate surface. Next,
N-maleimidecaproyloxy succinimide (hereinafter abbreviated as EMCS) was
dissolved in a 1:1 (volume ratio) solvent mixture of dimethyl sulfoxide
and ethanol to obtain a final concentration of 0.3 mg/ml to obtain an
EMCS solution. As EMCS, N-(6-maleimidecaproyloxy) succinimide
manufactured by Dojindo Laboratories was used.

[0114]The baked glass substrate was left standing and cooled and immersed
in the prepared EMCS solution at room temperature for 2 hours. By this
treatment, the amino group introduced to the surface of the substrate by
the silane coupling agent reacted with the succinimide group in the EMCS
to thereby introduce the maleimide group to the surface of the glass
substrate. The glass substrate picked up from the EMCS solution was
cleaned by using the above-mentioned solvent mixture in which the EMCS
was dissolved. The glass substrate was further cleaned by ethanol and
dried in a nitrogen gas atmosphere.

[0115](3-3. Probe DNA)

[0116]The microorganism detection probe prepared in the item (1.
Preparation of probe DNA) of Example 1 was dissolved in pure water. The
solution was dispensed such that the final concentration (at ink
dissolution) was 10 μM. Then, the solution was freeze-dried to remove
water.

[0117](3-4. DNA Discharge by BJ Printer and Bonding to Substrate)

[0118]An aqueous solution containing 7.5-wt % glycerin, 7.5-wt %
thiodiglycol, 7.5-wt % urea, and 1.0-wt % Acetylenol EH (manufactured by
Kawaken Fine Chemicals Co., Ltd.) was prepared. Each of the 4 probes
(Table 1) prepared in advance was dissolved in the solvent mixture at a
predetermined concentration. An ink tank for an inkjet printer (trade
name: BJF-850, manufactured by Canon Inc.) is filled with the resultant
DNA solution and attached to the printhead. Note that the inkjet printer
used here was modified in advance to allow printing on a flat plate. When
a printing pattern is input in accordance with a predetermined file
creation method, about 5-picoliter of a DNA solution can be spotted at a
pitch of about 120 μm. Subsequently, the printing operation was
executed for one glass substrate by using the modified inkjet printer to
prepare an array. After confirming that printing was reliably executed,
the glass substrate was left standing in a humidified chamber for 30
minutes to make the maleimide group on the glass substrate surface react
with the thiol group at the nucleic acid probe terminal.

[0119](3-5. Cleaning)

[0120]After reaction for 30 minutes, the DNA solution remaining on the
surface was cleaned by using a 10-mM phosphate buffer (pH 7.0) containing
100-mM NaCl, thereby obtaining a DNA chip in which single-stranded DNAs
were immobilized on the glass substrate surface.

Example 2

Detection of Candida glabrata

[0121]In this example, the detection of a microorganism using a two-step
PCR method is described.

[0126]The collected DNA of the microorganism (Candida glabrata) was
subjected to agarose electrophoresis and absorbance determination at
260/280 nm to examine the qualities of the DNA (i.e., the amount of
low-molecular weight nucleic acid as a contaminant and the degree of
decomposition) and the collected amount of the DNA. In this example,
about 10 μg of DNA was collected, while the degradation of DNA and the
contamination of ribosomal RNA were not recognized. The collected DNA was
dissolved in a TE buffer to a final concentration of 50 ng/μl and then
used in the example described below.

[0127](2. Amplification and Labeling of Specimen)

[0128](2-1. Amplification of Specimen: 1st PCR)

[0129]An amplification (1st PCR) reaction of microorganism DNA as an
analyte is shown in Table 1 below. The amplification reaction employed
the primer set shown in the item (2-1. Culture of microorganism and DNA
extraction therefrom) of Example 1, as described above.

[0130]The amplification reaction was carried out by a
commercially-available thermal cycler with the reaction solution of the
above-mentioned composition in accordance with the protocol illustrated
in FIG. 1.

[0131]After completing the reaction, the amplified product was purified
using a purification column (QIAquick PCR Purification Kit manufactured
by QIAGEN), followed by the quantitative assay of the amplified product.

[0132](2-2. Labeling Reaction: 2nd PCR)

[0133]A reaction solution of the composition shown in Table 5 was
subjected to an amplification reaction with a commercially-available
thermal cycler in accordance with the protocol illustrated in FIG. 2. The
amplification reaction employed the primer set shown in the item (2-2.
Preparation of PCR primer for labeling) of Example 1, as described above.

[0134]After completing the reaction, the primer was purified using a
purification column (QIAquick PCR Purification Kit manufactured by
QIAGEN) to obtain a labeled specimen.

[0135](3. Hybridization)

[0136]Detection reaction was performed using the DNA chip prepared in the
item (3. Preparation of DNA chip) of Example 1 and the labeled specimen
prepared in the item (2. Amplification and labeling of specimen) of
Example 2.

[0137](3-1. Blocking of DNA Chip)

[0138]Bovine serum albumin (BSA, Fraction V: manufactured by Sigma) was
dissolved in a 100-mM NaCl/10-mM phosphate buffer such that a 1 wt %
solution was obtained. Then, the DNA chip prepared in the item (3.
Preparation of DNA chip) of Example 1 was immersed in the solution at
room temperature for 2 hours to execute blocking. After completing the
blocking, the chip was cleaned using a washing solution as described
below, rinsed with pure water and hydro-extracted by a spin dryer.

[0141]A drained DNA chip was set on a hybridization device (Hybridization
Station, manufactured by Genomic Solutions Inc.) and a hybridization
reaction was then carried out using a hybridization solution shown in
Table 6 below in accordance with the protocol illustrated in FIG. 3.

[0143]The DNA chip after the above-mentioned hybridization reaction was
subjected to a fluorescent assay using a DNA-chip fluorescence detector
(GenePix 4000B, manufactured by Axon Co., Ltd.). Consequently, Candida
glabrata could be detected with good reproducibility and sufficient
signal intensity. The measurement results of Candida glabrata are shown
in Table 7 below.

[0144]As shown in Table 7 above, the first to fourth probes for Candida
glabrata shown in Table 1 expressed specific hybridization and it was
confirmed that there was a nucleic acid sequence in the DNA extract from
the microorganism culture medium which has the same sequence as that of
the ITS region of the DNA of Candida glabrata. Therefore, it can be
concluded that the DNA chip will allow the detection of Candida glabrata.

[0145](5. Results)

[0146]As is evident from the above-mentioned description, the
above-mentioned examples allowed the preparation of a DNA chip on which a
probe set, which was able to detect Candida glabrata, was immobilized.
Further, the use of the DNA chip allowed the identification of a
pathogenic fungus, so the problems of the DNA probe derived from a
microorganism was solved. In other words, the oligonucleotide probe can
be chemically produced in large amounts, while the purification or
concentration thereof can be controlled. For the purpose of classifying
the species of microorganisms, a probe set capable of collectively
detecting the same fungus species could be provided. In addition,
according to the embodiment, the base sequence of the ITS region on the
DNA sequence of the pathogenic fungus can be sufficiently detected and
thus the presence of the pathogenic fungus can be effectively determined
with high accuracy.

Example 3

Experiment of other Fungi

[0147]This example describes that strong hybridization cannot be detected
on any of fungi other than Candida glabrata when the DNA chip prepared in
Example 1 is employed.

[0148](1. Extraction of DNA (Model Specimen))

[0149](1-1. Culture of Microorganism and DNA Extraction Therefrom)

[0150]The following fungal strains were cultured in a manner similar to
Example 2 and the DNA thereof was then extracted and purified. The
deposit number of each fungus strain is represented in the parentheses
after the name of the fungus.

[0152]The collected DNAs of the respective fungi were subjected to the
assay of the collected amount thereof as described in Example 2. The
collected DNA was dissolved in a TE buffer to have a final concentration
of 50 ng/μl and then used in the following example.

[0153](2. Amplification and Labeling of Specimen)

[0154](2-1. Amplification of Specimen: 1st PCR)

[0155]An amplification reaction of microorganism DNA to be provided as a
specimen was carried out in a manner similar to that of the item (2-1.
Amplification of specimen: 1st PCR) of Example 2, as described above.
After completing the reaction, the amplified product was purified using a
purification column (QIAquick PCR Purification Kit manufactured by
QIAGEN), followed by the quantitative assay of the amplified product.

(2-2. Labeling Reaction: 2nd PCR)

[0156]A labeling reaction was carried out using the amplified product
obtained in the item (2-1. Amplification of specimen: 1st PCR) in a
manner similar to that of the item (2-2. Labeling reaction: 2nd PCR) of
Example 2, as described above. After completing the reaction, the labeled
product was purified using a purification column (QIAquick PCR
Purification Kit manufactured by QIAGEN), thereby obtaining a labeled
specimen.

(3. Hybridization)

[0157]A detection reaction was carried out using the DNA chip manufactured
in the item (3. Manufacture of DNA chip) of Example 1, as described above
and the labeled specimen prepared in the item (2. Amplification and
labeling of specimen) in a manner similar to the item (3. Hybridization)
of Example 2, as described above.

[0158](4. Detection of Microorganism (Fluorescent Assay))

[0159]A fluorescent assay was carried out in a manner similar to the item
(4. Detection of microorganism (fluorescent assay)) of Example 2 as
described above. The measurement results of the respective fungal species
are shown in Tables 8 to 32 below.

[0160]As shown in Table 8 above, in the probe set for Candida glabrata
shown in Table 1, it is evident that any specific hybridization does not
occur in all probes, compared with the experimental results of Table 7
above. It can be concluded that the DNA chip scarcely cross-hybridize to
Candida albicans.

[0161]As shown in Table 9 above, in the probe set for Candida glabrata
shown in Table 1, it is evident that any specific hybridization does not
occur in all probes, compared with the experimental results of Table 7
above. It can be concluded that the DNA chip scarcely cross-hybridize to
Candida dubliniensis.

[0162]As shown in Table 10 above, in the probe set for Candida glabrata
shown in Table 1, it is evident that any specific hybridization does not
occur in all probes, compared with the experimental results of Table 7
above. It can be concluded that the DNA chip scarcely cross-hybridize to
Candida guilliermondii.

[0163]As shown in Table 11 above, in the probe set for Candida glabrata
shown in Table 1, it is evident that any specific hybridization cannot
occur in all probes, compared with the experimental results of Table 7
above. It can be concluded that the DNA chip scarcely cross-hybridize to
Candida intermedia.

[0164]As shown in Table 12 above, in the probe set for Candida glabrata
shown in Table 1, it is evident that any specific hybridization cannot
occur in all probes, compared with the experimental results of Table 7
above. It can be concluded that the DNA chip scarcely cross-hybridize to
Candida kefyr.

[0165]As shown in Table 13 above, in the probe set for Candida glabrata
shown in Table 1, it is evident that any specific hybridization does not
occur in all probes, compared with the experimental results of Table 7
above. It can be concluded that the DNA chip scarcely cross-hybridize to
Candida krusei.

[0166]As shown in Table 14 above, in the probe set for Candida glabrata
shown in Table 1, it is evident that any specific hybridization does not
occur in all probes, compared with the experimental results of Table 7
above. It can be concluded that the DNA chip scarcely cross-hybridize to
Candida lusitaniae.

[0167]As shown in Table 15 above, in the probe set for Candida glabrata
shown in Table 1, it is evident that any specific hybridization does not
occur in all probes, compared with the experimental results of Table 7
above. It can be concluded that the DNA chip scarcely cross-hybridize to
Candida parapsilosis.

[0168]As shown in Table 16 above, in the probe set for Candida glabrata
shown in Table 1, it is evident that any specific hybridization does not
occur in all probes, compared with the experimental results of Table 7
above. It can be concluded that the DNA chip scarcely cross-hybridize to
Candida tropicalis.

[0169]As shown in Table 17 above, in the probe set for Candida glabrata
shown in Table 1, it is evident that any specific hybridization does not
occur in all probes, compared with the experimental results of Table 7
above. It can be concluded that the DNA chip scarcely cross-hybridize to
Trichosporon cutaneum.

[0170]As shown in Table 18 above, in the probe set for Candida glabrata
shown in Table 1, it is evident that any specific hybridization does not
occur in all probes, compared with the experimental results of Table 7
above. Consequently, It can be concluded that the DNA chip scarcely
cross-hybridize to Trichosporon asahii.

[0171]As shown in Table 19 above, in the probe set for Candida glabrata
shown in Table 1, it is evident that any specific hybridization does not
occur in all probes, compared with the experimental results of Table 7
above. It can be concluded that the DNA chip scarcely cross-hybridize to
Cryptococcus neoformans.

[0172]As shown in Table 20 above, in the probe set for Candida glabrata
shown in Table 1, it is evident that any specific hybridization does not
occur in all probes, compared with the experimental results of Table 7
above. It can be concluded that the DNA chip scarcely cross-hybridize to
Aspergillus fumigatus.

[0173]As shown in Table 21 above, in the probe set for Candida glabrata
shown in Table 1, it is evident that any specific hybridization does not
occur in all probes, compared with the experimental results of Table 7
above. It can be concluded that the DNA chip scarcely cross-hybridize to
Aspergillus niger.

[0174]As shown in Table 22 above, in the probe set for Candida glabrata
shown in Table 1, it is evident that any specific hybridization does not
occur in all probes, compared with the experimental results of Table 7
above. It can be concluded that the DNA chip scarcely cross-hybridize to
Epidermophyton floccosum.

[0175]As shown in Table 23 above, in the probe set for Candida glabrata
shown in Table 1, it is evident that any specific hybridization does not
occur in all probes, compared with the experimental results of Table 7
above. It can be concluded that the DNA chip scarcely cross-hybridize to
Arthroderma otae.

[0176]As shown in Table 24 above, in the probe set for Candida glabrata
shown in Table 1, it is evident that any specific hybridization does not
occur in all probes, compared with the experimental results of Table 7
above. It can be concluded that the DNA chip scarcely cross-hybridize to
Arthroderma gypseum.

[0177]As shown in Table 25 above, in the probe set for Candida glabrata
shown in Table 1, it is evident that any specific hybridization does not
occur in all probes, compared with the experimental results of Table 7
above. It can be concluded that the DNA chip scarcely cross-hybridize to
Arthroderma benhamiae.

[0178]As shown in Table 26 above, in the probe set for Candida glabrata
shown in Table 1, it is evident that any specific hybridization does not
occur in all probes, compared with the experimental results of Table 7
above. It can be concluded that the DNA chip scarcely cross-hybridize to
Trichophyton rubrum.

[0179]As shown in Table 27 above, in the probe set for Candida glabrata
shown in Table 1, it is evident that any specific hybridization does not
occur in all probes, compared with the experimental results of Table 7
above. It can be concluded that the DNA chip scarcely cross-hybridize to
Trichophyton tonsurans.

[0180]As shown in Table 28 above, in the probe set for Candida glabrata
shown in Table 1, it is evident that any specific hybridization does not
occur in all probes, compared with the experimental results of Table
above. It can be concluded that the DNA chip scarcely cross-hybridize to
Trichophyton verrucosum.

[0181]As shown in Table 29 above, in the probe set for Candida glabrata
shown in Table 1, it is evident that any specific hybridization does not
occur in all probes, compared with the experimental results of Table
above. It can be concluded that the DNA chip scarcely cross-hybridize to
Trichophyton violaceum.

[0182]As shown in Table 30 above, in the probe set for Candida glabrata
shown in Table 1, it is evident that any specific hybridization does not
occur in all probes, compared with the experimental results of Table 7
above. It can be concluded that the DNA chip scarcely cross-hybridize to
Arthroderma vanbreuseghemii.

[0183]As shown in Table 31 above, in the probe set for Candida glabrata
shown in Table 1, it is evident that any specific hybridization does not
occur in all probes, compared with the experimental results of Table 7
above. It can be concluded that the DNA chip leads low crow hybridization
to Arthroderma incurvatum.

[0184]As shown in Table 32 above, in the probe set for Candida glabrata
shown in Table 1, it is evident that any specific hybridization does not
occur in all probes, compared with the experimental results of Table 7
above. It can be concluded that the DNA chip scarcely cross-hybridize to
Trichophyton interdigitale.

[0185]As descried above, according to Tables 8 to 32, the DNA chip having
a low possibility of accidentally detecting any fungus other than Candida
glabrata, on which the probe set for Candida glabrata was immobilized,
could be prepared. The use of the DNA chip allows the detection of
Candida glabrata. Besides, a probe set which can detect Candida glabrata
while distinguishing it from other fungus species could be provided.
Further, it has been described that the DNA chip produced in Example 1
can only specifically hybridize with Candida glabrata but not hybridize
with any of other fungus species. Therefore, in a DNA chip which is
prepared as a combination of the probe for Candida glabrata as described
in Example 1 and a probe for other fungus species designed by the same
idea as that of the former, a probe set can be provided so that it can
not only detect Candida glabrata but also selectively detect Candida
glabrata even in a specimen containing a mixture of Candida glabrata with
other fungus species.

[0186]The present invention is not limited to the above embodiments and
various changes and modifications can be made within the spirit and scope
of the present invention. Therefore to apprise the public of the scope of
the present invention, the following claims are made.

[0187]While the present invention has been described with reference to
exemplary embodiments, it is to be understood that the invention is not
limited to the disclosed exemplary embodiments. The scope of the
following claims is to be accorded the broadest interpretation so as to
encompass all such modifications and equivalent structures and functions.

[0188]This application claims the benefit of Japanese Patent Application
No. 2007-128625, filed May 14, 2007, which is hereby incorporated by
reference herein in its entirety.